Master of Science, The Ohio State University, 2006, Graduate School

Committee: Not Provided (Other) Subjects:

Bachelor of Science (BS), Ohio University, 2024, Chemistry

The mitochondria-associated membrane (MAM) is a specialized subcellular compartment that provides a dynamic crosstalk between the endoplasmic reticulum (ER) and mitochondria. MAMs are a vital location for cellular processes such as calcium signaling, apoptosis, autophagy, and the response to stress. This thesis investigates the expression levels and spatial location of the MAM proteins ATG14, GRP75, MFN2, ERLIN1, ERLIN2, and VDAC1 in human keratinocytes (HaCaT cells) following acute and chronic simulated solar ultraviolet (sUV) irradiation, which has not yet been researched. The focus is on characterizing the changes in expression and spatial localization of these proteins, which are critical for maintaining cellular homeostasis and managing stress responses. HaCaT cells were exposed to sUV radiation mimicking natural sunlight for a single exposure (acute) and for three consecutive days (chronic), with subsequent analysis of protein responses at various time intervals post-exposure. Western blot analysis revealed that MFN2 and GRP75, proteins involved in structural and functional integrity of the MAM through their role in ER-mitochondria tethering, displayed a notable decrease in expression 6 hours post acute exposure. After chronic exposure, there was a significant upregulation of ATG14, ERLIN1, ERLIN2, and VDAC1, suggesting an adaptive cellular response to prolonged sUV stress. All targeted MAM proteins demonstrated a tendency to aggregate around the nucleus following sUV irradiation. This spatial reorganization may serve as an adaptive strategy to enhance mitochondrial-nuclear communication, potentially facilitating efficient energy transfer necessary for DNA repair and cellular survival during stress conditions. This study provides insights into stress response mechanisms within the MAM after sUV radiation.

Committee: Lauren McMills (Advisor); Shiyong Wu (Advisor); Veronica Bahamondes Lorca (Advisor) Subjects: Biochemistry

Bachelor of Science (BS), Ohio University, 2024, Chemistry

In a world where antibiotic resistance is on the rise, the pursuit of novel bacterial drug targets is increasingly important. The T-box riboswitch mechanism, which is involved in regulating the ability for Gram-positive bacteria to make proteins, is a promising target for a novel antibiotic. This study attempts to further understand this mechanism of transcription antitermination and termination in terms of the cofactors that modulate its outcome. The effects of two polyamine molecules, spermine and spermidine, on the riboswitch were analyzed using in-vitro transcription (IVT) assays and denaturing electrophoresis gels. The majority of this work focuses on optimizing the procedure of analysis, and this procedure was finally used to compare the transcription outcomes of the IVT assays that used the two different polyamines. The study concludes that spermine and spermidine differentially modulate the T-box riboswitch transcription outcomes, and that a hypothesized complex formation could have physiological significance in the bacterial cell. Further research is required to elucidate the reasoning behind this difference, but making slight changes to the procedure will likely help make this distinction.

Committee: Jennifer Hines (Advisor); Lauren McMills (Advisor) Subjects: Biochemistry; Chemistry

Bachelor of Science (BS), Ohio University, 2024, Chemistry

Due to the ever-growing health concern of antibiotic resistance, there is a need for novel drug development that can target bacteria in a way that is not resisted. In Gram positive bacteria, one of these potential targets is the T-box Riboswitch, which is a regulatory, non-coding region of RNA involved in amino acid regulation. Within the T-box Riboswitch is an antiterminator region, which dictates whether or not the downstream genes are transcribed. The genes that are regulated by the T-box riboswitch are directly involved in protein synthesis. The antiterminator is stabilized by the binding of uncharged tRNA, and when unbound forms the more stable terminator form, in which transcription is terminated. Drug design can be employed to target the antiterminator to prevent antitermination from occurring and cause the bacterial cell to die due to lack of protein synthesis. This thesis explores fragment-based computational docking studies to determine compounds that bind to the antiterminator with specificity and in regions that could potentially result in an inhibitory effect on antitermination. A compound library of 180 amino acid R groups were prepared and docked to the antiterminator as well as a control model without the tRNA-binding bulge region. Of the initial 180 amino acid R groups, 47 were determined to bind to the antiterminator in regions that may lead to inhibition with more specificity than in the control model. Additionally, 10 peptide links that were produced from these 47 compounds were further docked and each showed some level of binding to the regions of interest in the antiterminator. The results of this project indicated 47 amino acids that could potentially be used as building blocks for drug synthesis, in addition to 10 peptides that may produce an inhibitory effect on the antiterminator. Further studies can be performed on these compounds, such as fluorescence assays and transcriptional assays, to further te (open full item for complete abstract)

Committee: Jennifer V. Hines (Advisor) Subjects: Biochemistry; Chemistry

Doctor of Philosophy, The Ohio State University, 2024, Biochemistry Program, Ohio State

Mass spectrometry-based proteomics has reached a renaissance in the past 20 years, as acquisition software and methods have exponentially increased. Simultaneously, the demand for high-throughput tumor models has increased with the passing of the 2020 FDA Modernization Act, which removed the need for animal testing before clinical trials. 3-dimension tumor models, such as spheroids, are an attractive preclinical model, as they are relatively cheap to generate, easy to grow, and scalable. Spheroid models have traditionally been formed with one cell line, which greatly reduces the complexity of the model compared to actual patient tumors. Within this work, we sought to develop a coculture 3D tumor model, which incorporates fibroblast and colon cancer cell lines into spheroids, then profile the spheroid proteome with data-independent acquisition (DIA). Additionally, we have developed methodology to build target assays using parallel reaction monitoring (PRM) for both high-throughput models for applications in cancer and immunology. We have further optimized PRM methods on a compact, unit-resolution mass spectrometer for analyzing low-input samples. We have described detailed applications of proteomics for the tumor microenvironment, as well as low-abundant immune cell populations for use in immunological studies.

Committee: Amanda Hummon (Advisor); Heather Powell (Committee Member); Maria Mihaylova (Committee Member); Brian Searle (Committee Member) Subjects: Analytical Chemistry; Biochemistry; Biomedical Research; Chemistry

Master of Science, The Ohio State University, 2024, Molecular, Cellular and Developmental Biology

High throughput screening (HTS) has emerged as an efficient method for drug discovery. Unlike traditional screening methods, HTS is a highly sensitive and inexpensive tool that rapidly tests drug libraries to identify lead compounds for the treatment of human disease. Due to the high demand for therapeutic drugs, researchers must optimize new approaches in HTS. A limitation in performing HTS when studying protein-protein interactions is the large amount of protein required. To overcome this limitation, we have designed an approach based on the sequential addition of molecules to a single reaction well. We validated this approach by developing an assay to identify inhibitors of the interaction between the clathrin-coated vesicle adaptor protein CALM and the SNARE protein VAMP8. First, we developed a protocol to produce and isolate high yields of CALM-ANTH domain. Then we designed a fluorescence polarization assay to measure the binding constant of CALM-ANTH domain with VAMP8. Finally, we used the assay to screen over 10,000 compounds from an FDA-approved and commercially purchased library. We have identified a compound that can interfere with CALM-VAMP8 interaction named proflavine, a known DNA intercalating agent that commonly serves as an antiseptic for wound repair. Moreover, we validated a novel strategy for HTS that requires lower amounts of protein when compared with traditional HTS approaches. Our method yields similar results and allows the investigator to obtain direct information on a single compound basis. These studies may lead to increased efficiency of HTS and improved drug identification.

Committee: Emanuele Cocucci M. D., Ph. D. (Advisor); Richard Fishel Ph. D. (Committee Member); Blake Peterson Ph. D. (Committee Member); Anthony Brown Ph. D. (Committee Member) Subjects: Biochemistry; Pharmacology

Doctor of Philosophy, Case Western Reserve University, 2023, Pharmacology

Mitochondria form dynamic networks and need to maintain a delicate balance between fission and fusion to satisfy the cell's energetic and metabolic requirements. Fission is necessary to ensure mitochondria are properly distributed throughout the cell and to remove damaged mitochondrial components. The dysregulation of mitochondrial dynamics resulting in either an abnormally fragmented or interconnected mitochondrial network is associated with a variety of pathologies. Mutations in dynamin-related protein 1 (Drp1), the master regulator of mitochondrial fission, have been identified in patients presenting with severe neurological defects. Patient-derived fibroblasts exhibit hyperfused mitochondria, indicating mitochondrial dysregulation. Thus, these clinically relevant mutations impair Drp1 function, but the mechanism by which these mutations disrupt mitochondrial fission was undetermined. To address this lack of knowledge, the overarching objective of this research was to elucidate the specific Drp1 functional defects that are caused by these disease-associated mutations to better understand the relationship between impaired Drp1 function and disease. Drp1 self-assembles around the outer mitochondrial membrane (OMM) and, subsequently, hydrolyzes GTP which provides the mechanical force required to cleave apart the mitochondrion. The recruitment of Drp1 to the OMM is mediated in part by lipid interactions. A mitochondria-specific lipid, cardiolipin, promotes Drp1 self-assembly, enhances its GTPase activity, and is believed to facilitate membrane constriction. Disease-associated mutations in Drp1 are predominantly located within the GTPase and middle domains, which mediate its capabilities for hydrolysis and self-assembly, respectively. We have employed an ensemble of biochemical and EM-based techniques to investigate the impact of these mutations on the self-assembly, lipid recognition, and enzymatic capabilities of Drp1. Ultimately, we have shown that even mutatio (open full item for complete abstract)

Committee: Jason Mears (Advisor); Marvin Nieman (Committee Chair); Phoebe Stewart (Committee Member); Danny Manor (Committee Member); Edward Yu (Committee Member) Subjects: Biochemistry; Biomedical Research; Biophysics; Molecular Biology

Doctor of Philosophy, The Ohio State University, 2023, Chemistry

Actin is a 42 kDa ATPase that polymerizes into filaments, which are brought together into larger structures by actin-binding proteins. Actin and its partners comprise the actin cytoskeleton, which is ubiquitous in nature and is required for cell processes such as migration, division, and endocytosis. Bacteria have evolved their own set of actin-binding proteins, that they use to reorganize a eukaryotic host's cytoskeleton for their own ends. In this work, we explore the mechanisms of actin interaction by the bacterial protein Salmonella invasion protein A (SipA), and the human plastin proteins PLS2 and PLS3. SipA is a toxin expressed by Salmonella enterica serovar Typhimurium (Salmonella), which is delivered into host intestinal cells during infection. SipA is essential for Salmonella to invade host cells in the epithelium, where it becomes engulfed into a specialized compartment called the Salmonella-containing vacuole (SCV). The C-terminal half of SipA (SipA-C) has potent actin-binding ability and was previously shown to protect actin filaments from depolymerization by host proteins. We solved a high-resolution cryo-EM structure of SipA-C bound to F-actin and characterized their interaction. We found that SipA-C binds actin with a Kd of 26 pM, protects filaments from severing and depolymerization by human actin depolymerizing factor (ADF) at low mole ratios to actin, increases filament stiffness, and increases its thermal stability. Each of these behaviors are caused by the unique binding mechanism of SipA-C's Arm2 domain which penetrates the interprotofilament space of F-actin, a binding mode previously only observed with small peptide toxins. The cell regulates pH during migration. The actin-binding proteins ADF and talin are known to be sensitive to pH. These proteins enhance the turnover of focal adhesions during cell migration at alkaline pH, generated by the membrane-bound Na+/H+ exchanger NHE1. We found that human plastin 2 (PLS2) is also sensitive to (open full item for complete abstract)

Committee: Dmitri Kudryashov (Advisor); Samir Ghadiali (Committee Member); Tom Magliery (Committee Member); Venkat Gopalan (Committee Member) Subjects: Biochemistry

Master of Science (MS), Wright State University, 2023, Biochemistry and Molecular Biology

Pomegranate is known to have antioxidant and prebiotic qualities that have been shown to promote the growth of beneficial bacteria while reducing inflammation in the gut. Inflammation in the gut is an issue that results in many health problems including obesity and colon cancer. In this study, an experimental group received a daily pomegranate supplement for three weeks where a control group did not receive any supplement. After sequencing gDNA isolated from fecal samples from both before and after the trial period there was a significant difference between the two groups. The largest amount of variability is attributed to the individual the sample came from. However, pomegranate did significantly contribute to a change in the gut microbiome. Multiple different genera were changed between the pre and post-pomegranate trial samples. Two of these genera, Limosilactobacillus and Enterococcus, are both lactic acid bacteria. Lactic acid bacteria are known to have anti-inflammatory qualities within the gut. Other genera, including Collinsella are reduced during the trial period. Collinsella promotes inflammation in the gut that can lead to many intestinal diseases including irritable bowel syndrome and ulcerative colitis. Overall, this study shows that pomegranate consumption results in a significant change to the gut microbiome by promoting anti-inflammatory bacteria while reducing pro-inflammatory bacteria. The changes in the gut by pomegranate consumption helps protect against inflammatory associated diseases including type 2 diabetes and irritable bowel disease.

Committee: Oleg Paliy Ph.D. (Advisor); Weiwen Long Ph.D. (Committee Member); Kwang-Jin Cho Ph.D. (Committee Member) Subjects: Biochemistry; Microbiology; Molecular Biology

Doctor of Philosophy, The Ohio State University, 2023, Chemical Engineering

Microtubules are essential cytoskeletal polymers that are critically involved in diverse cellular processes ranging from intracellular transport to cell division and motility. To support these myriad functions, microtubules assemble chemically and morphologically diverse arrays. The axonemal microtubules in cilia and flagella are abundantly modified with chemically diverse posttranslational modifications that are important for cilia and flagella biogenesis, maintenance, motility, and signaling. Glycylation, the addition of variable numbers of glycines to internal glutamate residues in tubulin, is an understudied tubulin post-translational modification found almost exclusively in axonemal microtubules. Glycylation is mediated by enzymes of the Tubulin Tyrosine Ligase-Like (TTLL) family, of which there are three – TTLL3, 8, and 10 – in mammals. In cellulo and in vivo studies suggest that this modification is important for the biogenesis and stability of primary and motile cilia, and that the loss of glycylation may lead to male subfertility and increased cellular proliferation. However, the immense biochemical complexity of the microtubule cytoskeleton and its associated proteins makes it difficult to study the effects of individual tubulin post-translational modifications on specific microtubule effectors. Thus, in vitro reconstitution assays are necessary to elucidate the precise nature of these interactions. In this work, we characterize the TTLL8 and TTLL10 glycylase enzymes. Using tandem mass-spectroscopy we show that, unlike TTLL3 which preferentially monoglycylates β-tubulin tails, TTLL8 adds monoglycines at multiple internal glutamate positions on both α- and β-tubulin tails. TTLL10 elongates these monoglycines, generating long poly-glycine chains on both α- and β-tubulin. Using microscopy-based assays we show that monoglycylation is required for efficient TTLL10 binding to microtubules, and that polyglycylation suggests a self-limiting mechan (open full item for complete abstract)

Committee: Antonina Roll-Mecak (Advisor); David Wood (Advisor); Sarah Heissler (Committee Member); Chalmers Jeffrey (Committee Member); Eduardo Reategui (Committee Member); Stephen Osmani (Committee Member) Subjects: Biochemistry; Molecular Biology

Bachelor of Science (BS), Ohio University, 2023, Biological Sciences

The skin is the largest organ of the human body and has highly complex functions in the body. It serves an important role in protecting against chemical and physical factors, such as ultraviolet (UV) radiation from the sun. The UV radiation emitted from the sun is composed of UVA, UVB, and UVC wavelengths ranging from 200 nm to 400 nm. UVA is the portion having the longest wavelength (320 nm - 400 nm) and has the deepest penetration into the skin. UVB (280 nm - 320 nm) penetrates the skin only into the superficial layers. UVC (200 nm - 280 nm) is blocked by the atmosphere and does not reach the Earth's surface. When our skin is exposed to solar UV (sUV) radiation, many immediate and delayed reactions occur, having both positive and negative effects on the body. One particular effect of sUV exposure is the increase in reactive oxygen and nitrogen species from the activation of the nitric oxide synthases (NOSs). UVB induces rapid activation of the constitutive NOS (cNOSs), including endothelial NOS (eNOS) and neuronal NOS (nNOS). Upon activation, cNOSs produce nitric oxide (NO·) or superoxide (O2·-), the two molecules that can react rapidly to form peroxynitrite (ONOO-). Previously we showed that cNOSs are involved in the apoptotic signaling pathway via translational regulation of IκB expression and NF-κB activation in human keratinocyte HaCaT cells post-UVB exposure. To further study the role of sUV-induced activation of cNOS in skin cells, DNA damage and repair, inflammation, and photoaging were analyzed. An in vitro model was established using fibroblasts isolated from cNOS knock-out mice. In this study, we provide evidence that cNOSs are also involved in DNA damage repair post-sUV, the processing of MMP2, and the production of IL6.

Committee: Shiyong Wu (Advisor); Veronica Bahamondes Lorca (Advisor); Soichi Tanda (Advisor) Subjects: Biochemistry; Biology; Biomedical Research

Bachelor of Science (BS), Ohio University, 2023, Chemistry

There is a need for a skin model which combines the natural physiology of skin and uses less mice. Natural physiology is obtained using fresh, intact skin explants sourced from living organisms such as humans or mice. This work focused on the standardization and characterization of an in vitro mouse skin explant model for studying ultraviolet (UV) damage and repair mechanisms in skin. In this study, we established a protocol to use skin explants from discarded mice after euthanasia; these skin explants are cuts of intact dermal and epidermal skin suspended in cell culture medium and maintained in vitro. Our tissue explants maintain in vivo skin's characteristics and physiological responses for short periods of incubation time, but use less mice. In addition, our model allows us to study DNA damage and repair and still analyze different incubation periods post-sUV.

Committee: Lauren McMills Dr. (Advisor); Verónica Bahamondes Lorca Dr. (Advisor); Shiyong Wu Dr. (Advisor) Subjects: Biochemistry; Chemistry

Doctor of Philosophy, Case Western Reserve University, 2023, Chemistry

The emergence of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which is responsible for the COVID-19 pandemic has highlighted the need for rapid characterization of the viral mechanisms responsible for cellular pathogenesis. Viral untranslated regions (UTRs) represent conserved genomic elements that contribute to such mechanisms, and that are considered to be targets for therapeutic intervention. Details of the structures of most coronavirus (CoV) UTRs are not available, however. Experimental approaches are needed to allow for the facile generation of high-quality viral RNA tertiary structural models, which can facilitate comparative mechanistic and drug discovery efforts. By integrating experimental and computational techniques, we herein report the efficient characterization of conserved RNA structures within the 5'UTR of the human coronavirus OC43 (HCoV-OC43) genome, a lab tractable model coronavirus. Evidence is provided that the 5'UTR folds into a secondary structure with well-defined stem loops (SLs) as determined by chemical probing and direct detection of hydrogen bonds by NMR spectroscopy. We combine experimental base-pair restraints with global structural information from SAXS to generate a 3D model that reveals that SL1-4 adopts a topologically constrained structure wherein stem loops 3 and 4 co-axially stack. Co-axial stacking is mediated by short linker nucleotides and allows stem loops 1-2 to sample different co-joint orientations by pivoting about the SL3,4 helical axis. To evaluate the functional relevance of the SL3,4 co-axial helix, luciferase reporter constructs harboring the HCoV-OC43 5'UTR were engineered to contain mutations designed to abrogate co-axial stacking. The results reveal that the SL3,4 helix intrinsically represses translation efficiency since the destabilizing mutations correlate with increased luciferase expression relative to wild-type without affecting reporter mRNA levels. We also show that the nucleocapsid (open full item for complete abstract)

Committee: Blanton Tolbert (Advisor); Fu-Sen Liang (Committee Chair); Thomas Gerken (Committee Member); Robert Salomon (Committee Member); Divita Mathur (Committee Member) Subjects: Biochemistry; Chemistry

PhD, University of Cincinnati, 2022, Arts and Sciences: Chemistry

Recently, mechanochemistry has been a bustling industry as one of the leading alternative methodologies used to transform chemical reactions through mechanical impact. More specifically, soft mechanochemistry, a biomimetric strategy developed to understand macromolecular systems under the influence of mechanical force, has surged in its infancy in understanding forces that change structure and biophysical properties of biologically significant molecular components. This dissertation demonstrates the measuring capacity of soft mechanochemistry to investigate rheology and functional outcomes in mechanically treated proteins of unique size and function. To mimic the mechanical energies present in proteins' microenvironment, a modified ball mill containing several force variables such as oscillation frequency, milling temperature, and ball material was used. This work reveals for select proteins that small transformations to secondary structure elements and biological function can occur at specified frequencies that eliminates and/ or disrupts protein thermodynamic stability forces in a unique way. Our mechanochemical modifications allow control of destabilizing forces that effect the biological properties of proteins, in which we are able to detect the degree of force that induces molecular conformational transitions in protein unfolding. The mutual interactions of the biochemical properties and structural features of RNase T1, BSA, and CaLB reported here are important in soft mechanochemistry for interdisciplinary research in protein biomechanics and for the design and the development of new methods in cancer research for investigating proteins under mechanical stress.

Committee: James Mack Ph.D. (Committee Member); Ryan White Ph.D. (Committee Member); Balasubrahmanyam Addepalli Ph.D. (Committee Member) Subjects: Biomechanics

PhD, University of Cincinnati, 2022, Medicine: Molecular Genetics, Biochemistry, & Microbiology

Bone Morphogenetic Proteins (BMPs) are the largest subgroup of the Transforming Growth Factor ß (TGFß) superfamily, one of the fundamental protein signaling pathways in biology. BMPs are involved in regulating numerous biological functions, with a particular focus on development, immune modulation, cell homeostasis and wound healing. When dysregulated, aberrant BMP signaling can indue a host of developmental and autoimmune disorders, as well as many different cancers. Given the wide array of biological functions BMPs regulate, precise regulation of signaling is a key component of their biology. Mechanistically, these secreted dimeric signaling proteins function by forming complex with two type 1 and two type 2 serine/threonine kinase receptors on the cell surface to drive signaling by intracellular SMAD proteins. Accordingly, the regulation of these potent signaling molecules in the extracellular space is a vital area of study. The purpose of the work outlined in this thesis is to explore certain understudied mechanisms of extracellular modulation of BMP signaling. We particularly focused on studying these mechanisms not in isolation, but rather as they actually exist in nature, as part of a complex environment with many competing biomolecules. We present studies contrasting related protein antagonists with different function in an attempt to gain insight into the key components needed for BMP inhibition. In addition, we explore a newly discovered interaction between the BMP and Wnt signaling pathways, where BMP ligands may directly antagonize canonical Wnt signaling. Lastly, we describe a procedure for the production of artificial BMP heterodimeric signaling molecules using chains with differential activity with respect to receptor preference, antagonist targeting, and affinity to the extracellular matrix. These asymmetrical signaling molecules were then used to isolate the key components of biological function across multiple different experimental systems. Overal (open full item for complete abstract)

Committee: Thomas Thompson Ph.D. (Committee Member); Rhett Kovall Ph.D. (Committee Member); Aaron Zorn Ph.D. (Committee Member); James Wells Ph.D. (Committee Member); Michael Tranter Ph.D. (Committee Member) Subjects: Biochemistry

Master of Engineering, Case Western Reserve University, 2022, Biomedical Engineering

Mucin-type O-glycosylation is an abundant yet understudied post-translational modification of proteins in metazoans, initiated by the polypeptide N-acetylgalactosamine-transferases (GalNAc-Ts) of which twenty are known in humans. O-glycosylation is involved in many biological processes including intracellular signaling and cell-cell interactions. GalNAc-T mutations contribute to several disease states such as cancer. Previous characterization of the GalNAc-Ts has revealed unique preferences for peptide sequence and prior substrate glycosylation. Herein, another paradigm by which the GalNAc-Ts select peptide targets is investigated: flanking substrate charge. Twelve GalNAc-Ts have been characterized against substrates containing different N-/C-terminal charges, demonstrating unique and overlapping charge preferences. Electrostatic models, elevated ionic strength, and molecular dynamics simulations reveal that GalNAc-Ts selectivity is indeed modulated via charge-charge interactions. Michaelis-Menten kinetics revealed significant variation in kinetic parameters, particularly Vmax. Overall, this work reveals that charge-charge interactions are another important but previously overlooked factor that uniquely modulates GalNAc-T specificity.

Committee: Thomas Gerken (Advisor); Mei Zhang (Advisor); Xinning Wang (Committee Member); Ryan Arvidson (Committee Member); Sam Senyo (Committee Chair) Subjects: Biochemistry; Biomedical Engineering

Doctor of Philosophy, The Ohio State University, 2021, Biochemistry Program, Ohio State

Development and functionality of multicellular organisms relies on precise and strong adhesion between cells. Members of the cadherin superfamily of proteins are involved in calcium-dependent cell-cell adhesion in animals and have been shown to play vital roles in various relevant biological processes. The cadherin superfamily can be largely classified into three subfamilies: the classical cadherins, the non-clustered protocadherins, and the clustered protocadherins. The typical cadherin protein consists of a single-pass transmembrane domain, a cytoplasmic domain, and multiple non-identical extracellular cadherin (EC) repeats. These ECs are defined by their Greek-key fold and an EC linker region with highly conserved calcium-binding sites. The binding of calcium helps to provide the rigidity necessary for proper protein-protein interaction. Within this work I focus on cadherins responsible for mechanotransduction, both from the classical and non-clustered subfamilies. Adherens junctions are formed by classical members of the cadherin superfamily and provide strong adhesion between cells. Past experiments have determined that interactions between individual cadherins are weak and therefore the strength provided by epithelial cadherin (CDH1), the major cadherin of adherens junctions, must come about through the formation of cadherin complexes. These cadherin complexes are composed of multiple CDH1 molecules binding through trans- (ectodomains originating from adjacent cells) and cis-interactions (ectodomains originating from the same cell) as seen in x-ray crystal structures and cryo-electron tomography images. While most experiments have focused on single homodimers, the mechanical unbinding events of cadherin junctional complexes and their effect on the membrane and associated cytoplasmic proteins remains unexplored. My work on CDH1 junctional complexes and their associated proteins utilizes large-scale all-atom molecular dynamics (MD) simulations to probe the ad (open full item for complete abstract)

Committee: Marcos Sotomayor (Advisor); Mark Foster (Committee Member); Steffen Lindert (Committee Member); Charles Bell (Committee Member) Subjects: Biochemistry; Biomechanics; Biophysics

Doctor of Philosophy, The Ohio State University, 2021, Chemistry

Iron is the second most abundant metal on earth, and therefore it is not surprising that throughout the kingdoms of life iron is a vital metal in biological processes. A common form of iron that is conserved throughout all organisms is the iron-sulfur cluster. Examples include the [2Fe-2S] and [4Fe-4S] clusters that bind to a number of proteins with specific Fe-S cluster binding sites. Such sites normally consist of four cysteine residues, with occasional replacement of Cys with histidine or aspartate. Iron-sulfur clusters have been connected to several functions in the body, including metabolite synthesis, metabolism, DNA synthesis, biodegradation, and isomerization. The biosynthesis and trafficking of [2Fe-2S] clusters has been studied extensively within the mitochondria, but mysteries remain concerning the cellular chemistry of Fe-S clusters, particularly the cellular formation of [4Fe-4S] clusters and the relationship and possible trafficking of [2Fe-2S] clusters between mitochondria and cytosol of the cell. One of the largest families of proteins, the S-adenosylmethionine (SAM) family, binds a [4Fe-4S] cluster. Lipoyl synthase (LIAS) is a member of the radical S-adenosylmethionine (SAM) superfamily that catalyzes the final step of lipoic acid biosynthesis. The enzyme contains two [4Fe-4S] centers (reducing and auxiliary clusters) that promote radical formation and sulfur transfer, respectively. Most information concerning LIAS and its mechanism has been determined from prokaryotic enzymes. We have made progress in expressing, isolating and characterizing human LIAS, its reactivity, and evaluation of natural iron-sulfur (Fe-S) cluster reconstitution mechanisms. Cluster donation by a number of possible cluster donor proteins and heterodimeric complexes has also been evaluated. [2Fe-2S]-Cluster-bound forms of human ISCU and ISCA2 were found capable of reconstituting human LIAS such that complete product turnover was enabled for LIAS, as monitored via a liquid ch (open full item for complete abstract)

Committee: James Cowan (Advisor); Christine Thomas (Other); Claudia Turro (Committee Member); Ross Dalbey (Committee Member) Subjects: Biochemistry; Chemistry

Doctor of Philosophy, The Ohio State University, 2021, Chemistry

Cellular life is naturally orchestrated by the biochemical components of cells that include nucleic acids, lipids, carbohydrates, and proteins. Other vital components include co-factors such as metabolites and transition metals. All of these critical components coalesce inside the cell and function synchronously allowing for complex life. Many natural reactions essential for life require enzymes, several of which require metal co-factors for structural stability or to even mediate the catalytic reaction. These metalloenzymes allow for complex chemical processes, as they catalyze a host of biochemical reactions both efficiently and selectively, where the metal cofactor provides additional functionality to promote reactivity not readily achieved in their absence. The design of miniature enzymes has been explored for over the past sixty years in order to understand the fundamentals of enzyme catalysis and to design more efficient compounds. While mechanistic exploration has illuminated the details of certain catalytic systems, the development of smaller and more efficient catalysts is desired to meet industrial, medical, and environmental needs. Artificial metalloenzymes for glycosidase activity have been investigated previously, however, the development of selective small molecule and peptide-based metalloenzymes have been scarce. Herein, we describe the synthesis, enzymatic characterization, and explore the mechanism of small multi-nuclear copper catalysts as efficient metalloglycosidase mimics. These small molecules appear to mediate degradation of carbohydrate substrates via a CuII/I redox couple with a mainly metal-mediated reaction as very little of the reactive oxygen species (ROS) were diffusible. A synergistic effect is noted when comparing the dinuclear copper system to the mononuclear system in the presence of relevant co-reagents which aid in facilitating the redox reaction. As these complexes can be preferential for glucose and galactose-containing ca (open full item for complete abstract)

Committee: James Cowan (Advisor); Ross Dalbey (Committee Member); Claudia Turro (Committee Member) Subjects: Biochemistry; Chemistry; Inorganic Chemistry

Doctor of Philosophy, The Ohio State University, 2021, Physics

The ability to apply and measure high forces (≥10pN) on the nanometer scale is critical to the ongoing development of nanomedicine, molecular robotics, and the understanding of biological processes such as chromatin condensation, membrane deformation, and molecular motors [1] [2] [3]. Current force spectroscopy techniques rely on micron-sized handles to apply forces, which can limit applications within nanofluidic devices or cellular environments [4]. To overcome these limitations, I used deoxyribonucleic (DNA) origami to self-assemble a nanocaliper, building on previous designs[5] [6]. I characterize the nanocaliper via a short double-stranded (ds)DNA with each strand attached to opposite arms of the device, via device equilibrium state, output force, and dynamics, to understand the effects of sequence, vertex design, and strut length on the device properties. I also produce nucleosomes, hexasomes, and an alternate dsDNA, which were then measured in the device, yielding mechanistic insight into the free energy landscape of each. I measure forces greater than 20 pN applied by the device with a nanometer dynamic range and 1 to 10 pN/nm stiffness. These high performing characteristics which expand the capabilities of existing force spectroscopy techniques as well as those of DNA origami devices.

Committee: Michael Poirier (Advisor); Ralf Bundschuh (Committee Member); Carlos Castro (Committee Member); Ezekiel Johnston-Halperin (Committee Member) Subjects: Biochemistry; Biophysics; Nanoscience; Physics